Methods known for transformation of monocotyledons such as maize and rice, which are major grain crops, include electroporation, particle gun transformation, etc. However, these physical gene transfer methods have problems in that genes are introduced as multiple copies or are not inserted in an intact state, and the resulting transformed plants may often show abnormal in morphology and sterility.
Agrobacterium-mediated gene transfer is a method for plant transformation based on functions of Agrobacterium. A soil bacterium Agrobacterium (Agrobacterium tumefaciens) functions in such a manner that the T-DNA forming a part of its Ti (tumor-inducing) plasmid involved in the pathogenicity of the Agrobacterium is integrated into the genome of a plant when it infects the plant. Agrobacterium-mediated plant transformation is a method for introducing a desired gene into the genome of a plant through the above Agrobacterium function by constructing a transforming plasmid in which the T-DNA region of the Ti plasmid is replaced by the gene desired to be introduced into the plant genome and then using Agrobacterium prepared to carry the transforming plasmid in place of the Ti plasmid.
Agrobacterium-mediated gene transfer is universally used as a transformation method for dicotyledons. Although it has been understood that hosts of Agrobacterium are limited only to dicotyledons and Agrobacterium has no ability to infect monocotyledons (De Cleene, M. and De Ley, J., (1976) Bot. Rev., 42: 389-466), some attempts have been made to transform monocotyledons through Agrobacterium-mediated method (Grimsley, N., et al., (1987) Nature, 325: 177-179; Gould, J., et al., (1991) Plant Physiol., 95: 426-434; Mooney, P. A., et al., (1991) Plant Cell, Tissues and Organ Culture, 25: 209-218; Raineri, D. M., et al., (1990) Bio/technology, 8: 33-38). These study reports suggested that Agrobacterium-mediated gene transfer is also possible for Gramineae crops including rice, maize and wheat, but the reports failed to show persuasive results because these studies had a problem in reproducibility and were also insufficient for transgene confirmation (Potrycus, I., (1990) Bio/technology, 8: 535-542).
Chan et al. injured immature rice embryos, which had been cultured for 2 days in the presence of 2,4-D (2,4-dichlorophenoxyacetic acid), and then inoculated these embryos with Agrobacterium carrying genes for nptII and GUS in a medium containing suspension-cultured potato cells. They cultured the thus treated immature embryos on G418-containing medium to obtain regenerated plants from the induced calli. They confirmed the location of the GUS gene in the regenerated plants and their progeny by Southern analysis, and reported that the presence of the transgene was observed in both T0 generation of regenerated plants and their progeny (Chan, M-T., et al., (1993) Plant Mol. Biol., 22: 491-506). This result supports Agrobacterium-mediated transformation in rice, but the transformation efficiency was as low as 1.6%. Moreover, there was only one regenerated plant that showed normal growth, although 250 immature embryos were used for testing. Since laborious efforts are required to excise immature embryos of rice, such low transformation efficiency is not practical.
In recent years, it has been reported that stable and highly efficient transformation is also possible in monocotyledons including rice and maize when using a super-binary vector carrying a part of the virulence gene from super-virulent Agrobacterium (Hiei, Y., et al., (1994) The Plant Journal, 6: 271-282; Ishida, Y., et al., (1996) Nature Biotechnology, 14: 745-750). These reports suggest that Agrobacterium-mediated transformation not only allows stable and highly efficient transformation, but is also advantageous in that the resulting transformed plants have fewer mutations, and in that the introduced genes are low in copy number and are often in an intact state. Following success in rice and maize, further reports were issued for Agrobacterium-mediated transformation in other major grain crops, i.e., wheat (Cheng, M., et al., (1997) Plant Physiol., 115: 971-980), barley (Tingay, S., et al., (1997) Plant J., 11: 1369-1376) and sorghum (Zhao, Z.-Y., et al., (2000) Plant Mol. Biol., 44: 789-798).
Ishida et al. used maize inbred line A188 and A188-related inbred lines as materials to perform Agrobacterium-mediated transformation (Ishida, Y., et al., (1996) Nature Biotechnology, 14: 745-750). Thereafter, further reports were issued for Agrobacterium-mediated transformation in maize, each of which reports used A188 and A188-related hybrids (Deji, A., et al., (2000) Biochim. et Biophys. Acta, 1492: 216-220; Negrotto, D., et al., (2000) Plant Cell Reports, 19: 798-803; Nomura, M., et al., (2000) Plant J., 22: 211-221; Nomura, M., et al., (2000) Plant Mol. Biol., 44: 99-106; Taniguchi, M., et al., (2000) Plant Cell Physiol., 41: 42-48; Zhao, Z.-Y., et al., (2001) Mol. Breed., 8: 323-333; Frame, B. R., et al., (2002) Plant Physiol., 129: 13-22). Attempts which have been made to improve the efficiency of Agrobacterium-mediated maize transformation include: selection of transformed cells on N6 basal medium (Zhao, Z.-Y., et al., (2001) Mol. Breed., 8: 323-333); addition of AgNO3 and carbenicillin to culture medium (Zhao, Z.-Y., et al., (2001) Mol. Breed., 8: 323-333; Ishida, Y., et al., (2003) Plant Biotechnology, 20: 57-66); and addition of cysteine to co-culture medium (Frame, B. R., et al., (2002) Plant Physiol., 129: 13-22). Ishida et al. selected co-cultured immature maize embryos on a medium containing AgNO3 and carbenicillin to produce transformed plants from inbred lines H99 and W117, which are publicly available inbred other than A188, and also reported that this procedure improved the transformation efficiency in A188 (Ishida, Y., et al., (2003) Plant Biotechnology, 20: 57-66).
Singh and Chawla reported that immature wheat embryos expressing the GUS gene increased in number when mixed in a suspension of silicon carbide fibers (SCFs) with a vortex mixer for 2 to 3 minutes before being inoculated with Agrobacterium (Singh, N. and Chawla, S., (1999) Current Science, 76: 1483-1485). This is because the immature embryos were injured by SCFs. Other attempts to injure tissues before Agrobacterium inoculation include injuring with a particle gun (Bidney et al., 1992) and injuring by ultrasonication (Trick, H. N. and Finer, J. J., (1997) Transgenic Res., 6:329-336).
Various attempts have been made to improve the transformation efficiency in Agrobacterium-mediated maize transformation (Negrotto, D., et al., (2000) Plant Cell Reports, 19: 798-803; Zhao, Z.-Y., et al., (2001) Mol. Breed., 8: 323-333; Frame, B. R., et al., (2002) Plant Physiol., 129: 13-22; Ishida, Y., et al., (2003) Plant Biotechnology, 20: 57-66). However, the resulting effects are still low when compared to rice which is also a member of monocotyledons, so that further improvement in the transformation efficiency is desired not only for production of practical transformed maize plants, but also for confirmation of novel genes for their effect in maize. Moreover, in response to recent progress in genomics studies, the necessity of transformation has been increased for the purpose of gene function analysis. Thus, there will be a demand for efficient transformation systems.
Likewise, the development of a method achieving higher transformation efficiency in other monocotyledons and dicotyledons than that provided by current procedures is also useful in various instances where transformants are used.
All documents cited herein are incorporated herein by reference in their entirety.                Patent Document 1: Japanese Patent No. 2,649,287        Patent Document 2: Japanese Patent No. 3,329,819        Patent Document 3: Japanese Patent Public Disclosure No. 2000-342256        Patent Document 4: International Publication No. WO 95/06722        Non-patent Document 1: Bidney, D., et al., (1992) Plant Mol. Biol., 18: 301-313.        Non-patent Document 2: Chan, M-T., et al., (1993) Plant Mol. Biol., 22: 491-506.        Non-patent Document 3: Cheng, M., et al., (1997) Plant Physiol., 115: 971-980.        Non-patent Document 4: De Cleene, M. and De Ley, J., (1976) Bot. Rev., 42: 389-466.        Non-patent Document 5: Deji, A., et al., (2000) Biochim. et Biophys. Acta, 1492: 216-220.        Non-patent Document 6: Frame, B. R., et al., (2002) Plant Physiol., 129: 13-22.        Non-patent Document 7: Gould, J., et al., (1991) Plant Physiol., 95: 426-434.        Non-patent Document 8: Grimsley, N., et al., (1987) Nature, 325: 177-179.        Non-patent Document 9: Hiei, Y., et al., (1994) The Plant Journal, 6: 271-282.        Non-patent Document 10: Ishida, Y., et al., (1996) Nature Biotechnology, 14: 745-750.        Non-patent Document 11: Ishida, Y., et al., (2003) Plant Biotechnology, 20: 57-66.        Non-patent Document 12: Mooney, P. A., et al., (1991) Plant Cell, Tissues and Organ Culture, 25: 209-218.        Non-patent Document 13: Negrotto, D., et al., (2000) Plant Cell Reports, 19: 798-803.        Non-patent Document 14: Nomura, M., et al., (2000) Plant J., 22: 211-221.        Non-patent Document 15: Nomura, M., et al., (2000) Plant Mol. Biol., 44: 99-106.        Non-patent Document 16: Potrycus, I., (1990) Bio/technology, 8: 535-542.        Non-patent Document 17: Raineri, D. M., et al., (1990) Bio/technology, 8: 33-38.        Non-patent Document 18: Singh, N. and Chawla, S., (1999) Current Science, 76: 1483-1485.        Non-patent Document 19: Taniguchi, M., et al., (2000) Plant Cell Physiol., 41: 42-48.        Non-patent Document 20: Trick, H. N. and Finer, J. J., (1997) Transgenic Res., 6:329-336.        Non-patent Document 21: Tingay, S., et al., (1997) Plant J., 11: 1369-1376.        Non-patent Document 22: Zhao, Z.-Y., et al., (2000) Plant Mol. Biol., 44: 789-798.        Non-patent Document 23: Zhao, Z.-Y., et al., (2001) Mol. Breed., 8: 323-333.        Non-patent Document 24: Hoekema, A., et al., (1983) Nature, 303: 179-180        Non-patent Document 25: Komari, T. and Kubo, T., (1999) Methods of Genetic Transformation: Agrobacterium tumefaciens. In Vasil, I. K. (ed.), Molecular improvement of cereal crops, Kluwer Academic Publishers, Dordrecht, p. 43-82.        